The effect of PEG coating on in vitro cytotoxicity and in vivo disposition of topotecan loaded liposomes in rats

Amphoteric drugs encapsulated in PEGylated liposomes may not show superior therapeutic antitumor activity due to increased leakage rate of these drugs in presence of PEG-lipids. In order to investigate the effect of PEG coating on in vitro and in vivo characteristics of topotecan loaded liposomes, an amphoteric anticancer drug, PEGylated and conventional liposomes were prepared by lipid film hydration method. Various properties of the prepared nanoliposomes such as encapsulation efficiency, size, zeta potential, physical stability as well as the chemical stability of lactone form of topotecan, cytotoxicity and topotecan pharmacokinetics were evaluated. In vitro cytotoxic activity was evaluated on murine Lewis lung carcinoma (LLC) and human mammary adenocarcinoma (BT20) cells. Pharmacokinetic was evaluated in Wistar rats after i.v. injection of topotecan, formulated in PBS pH 7.4 or in conventional or in PEGylated liposomes. The conventional liposome (CL) formulation was composed of DSPC/cholesterol/DSPG (molar ratio; 7:7:3), while for PEGylated liposome the composition was DSPC/cholesterol/DSPG/DSPE-PEG(2000) (molar ratio; 7:7:3:1.28). The size of both liposomes was around 100 nm with polydispersity index of about 0.1. In comparison with free drug, liposomal topotecan showed more stability for topotecan lactone form in vitro. Compared to free topotecan, PEGylated and conventional liposomes improved cytotoxic effect of topotecan against the two cancer cell line studied. The results of pharmacokinetic studies in rats showed that both CL and PEGylated liposomal formulations increased the concentration of total topotecan in plasma, however, initial concentration and the values of AUC, MRT and t(1/2 beta) were much higher (P<0.001) for PEGylated liposomal drug than for conventional one or free drug. PEGylated liposome resulted in a 52-fold and 2-fold increases in AUC(0-infinity) compared with that of free topotecan and CL, respectively. These results indicated that PEG modified liposome might be an effective carrier for topotecan.     

Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity

Vinorelbine (VRL) is a particularly lipophilic member of the vinca alkaloids which, as a class of drugs, exhibit improved cytotoxicity and therapeutic activity through increased duration of exposure. Here, we describe and optimize a sphingomyelin/cholesterol (SM/Chol) liposome formulation of VRL to maximize in vivo drug retention, plasma circulation time, and therapeutic activity. VRL was efficiently encapsulated (>90%) into 100 nm liposomes using an ionophore-mediated loading method. VRL retention in SM/Chol liposomes after intravenous injection in mice was dependent on drug-to-lipid ratio (D/L), with higher D/L ratios exhibiting increased drug retention (0.3 > 0.2 > 0.1, wt/wt) and improved pharmacokinetics. Cryo-electron microscopic examination of a high D/L ratio formulation indicated that the intravesicular regions of these liposomes were electron dense compared with empty liposomes. The optimized, high D/L ratio SM/Chol VRL formulation showed promising activity against subcutaneous B16 melanoma tumors compared with VRL or SM/Chol formulations of vincristine or vinblastine. Finally, the stability of the formulation was excellent (<5% drug leakage, >99% intact VRL, no changes in liposome size after 1 year at 2-8 degrees C). The optimized drug retention properties of the SM/Chol formulation of VRL, combined with its promising antitumor activity and pharmaceutical stability, make this formulation an excellent candidate for future clinical development.     

离子载体A23187介导pH梯度法制备重酒石酸长春瑞滨长循环脂质体

目的:采用A23187制备重酒石酸长春瑞滨长循环脂质体,优化了处方工艺,并考察了含量、包封率、药脂比和体外释放等检测指标。方法:采用A23187介导的pH梯度法制备了重酒石酸长春瑞滨脂质体;用HPLC法检测了脂质体中重酒石酸长春瑞滨的含量和脂质(HSPC)的含量,考察了药脂比;采用阳离子交换树脂分离脂质体和游离药物,HPLC法检测包封率;以4 mmol.L-1NH4Cl-PBS(pH 7.4)为体外释放介质考察了脂质体的体外释放行为。结果:重酒石酸长春瑞滨脂质体包封率为96.1%,药脂比为1∶5(w/w);高药脂比有利于延长药物体外释放的时间。结论:采用A23187介导的pH梯度法制备重酒石酸长春瑞滨脂质体工艺可行、载药量大、包封率高;所建立体外释放的检测方法快速、准确。     

Liposome formulation of poorly water soluble drugs: optimisation of drug loading and ESEM analysis of stability

Liposomes due to their biphasic characteristic and diversity in design, composition and construction, offer a dynamic and adaptable technology for enhancing drug solubility. Starting with equimolar egg-phosphatidylcholine (PC)/cholesterol liposomes, the influence of the liposomal composition and surface charge on the incorporation and retention of a model poorly water soluble drug, ibuprofen was investigated. Both the incorporation and the release of ibuprofen were influenced by the lipid composition of the multi-lamellar vesicles (MLV) with inclusion of the long alkyl chain lipid (dilignoceroyl phosphatidylcholine (C24PC)) resulting in enhanced ibuprofen incorporation efficiency and retention. The cholesterol content of the liposome bilayer was also shown to influence ibuprofen incorporation with maximum ibuprofen incorporation efficiency achieved when 4 micromol of cholesterol was present in the MLV formulation. Addition of anionic lipid dicetylphosphate (DCP) reduced ibuprofen drug loading presumably due to electrostatic repulsive forces between the carboxyl group of ibuprofen and the anionic head-group of DCP. In contrast, the addition of 2 micromol of the cationic lipid stearylamine (SA) to the liposome formulation (PC:Chol - 16 micromol:4 micromol) increased ibuprofen incorporation efficiency by approximately 8%. However further increases of the SA content to 4 micromol and above reduced incorporation by almost 50% compared to liposome formulations excluding the cationic lipid. Environmental scanning electron microscopy (ESEM) was used to dynamically follow the changes in liposome morphology during dehydration to provide an alternative assay of liposome stability. ESEM analysis clearly demonstrated that ibuprofen incorporation improved the stability of PC:Chol liposomes as evidenced by an increased resistance to coalescence during dehydration. These finding suggest a positive interaction between amphiphilic ibuprofen molecules and the bilayer structure of the liposome.     

Formulation and characterization of azidothymidine-loaded liposomes

Entrapment efficiency (EE%) and in vitro stability of azidothymidine (AZT)-loaded hand-shaken multilamellar vesicles (MLVs), freeze and thaw vesicles (FATMLVs), and reverse phase evaporation vesicles (REVs) were compared. AZT entrapment in FATMLVs was further studied by varying initial lipid concentrations, drug concentration, and lipid composition. The results suggest that AZT entrapment is dependent on the aqueous volume entrapped within liposomes, and the interaction between the drug and liposomal bilayer may not be significant. Increasing the lipid concentration increases the liposomal entrapment of AZT but the encapsulation yield decreases above a lipid concentration of 30 μmol/mL. No significant difference was observed in EE% when the AZT concentration was varied from 5 to 20 mg/mL. The entrapment efficiency was highest (43.2%) for DSPC/CHOL/PS (molar ratio 6:3:3) vesicles but DSPC/CHOL/PS liposome formulations in a molar ratio of 4:3:3 or 4:5:1 and DSPC/CHOL/SA liposome formulations in a molar ratio of 4:5:1 were found to be more stable in vitro. In vitro drug release from liposomes was dependent on bilayer composition and the method of preparation.     

Free versus liposome-encapsulated lignocaine hydrochloride topical applications.

The influence of encapsulating lignocaine hydrochloride, in a liposomal delivery system on its release from a topical formulation was studied. Liposomes were prepared from a mixture of L-alpha-dipalmitoylphosphatidyl choline (DPPC) and cholesterol in different molar ratios with or without charge inducing agents. The swelling time affording maximum entrapment was tested from 0-48 h at 53 degrees C. The percentage of drug entrapped in liposomes was found to be charge dependent and more pronounced for negatively charged liposomes. The amount of drug entrapped increased by increasing the ratio of cholesterol. The selected formulations were evaluated in vivo using the pin prick method. The results revealed localized and prolonged activity of lignocaine contained in liposomes when compared with an equivalent conventional topical application.     

Characterization of calcitonin-containing liposome formulations for intranasal delivery

Calcitonin-containing liposome formulations were characterized to obtain information for evaluation of their feasibility in intranasal delivery. The parameters of liposomal charge characteristics, charge inducing agent concentration, calcitonin concentration and pH of the medium on the loading efficiency and leakage behaviour, and the chemical stability of calcitonin in liposomes were investigated. Results showed that the loading efficiency of calcitonin increased with increasing the added concentration of calcitonin. The magnitude of the loading efficiency due to the liposomal charge of negative, positive and neutral characteristics was in the order of negatively charged liposome > neutral liposome > positively charge liposome. The increase of molar ratio of phosphatidylserine in liposomes showed an increase of loading efficiency; while, the increase of molar ratios of stearylamine showed a decrease of loading efficiency. The loading efficiency at pH 7.4 was greater than that at pH 4.3. The leakage of positively charged liposomes was greater than that of neutral and negatively charged liposomes. The leakage at pH 4.3 was faster than that at pH 7.4. The leakage of positively charged liposomes increased as temperature increased. The chemical stability of calcitonin in both solution and liposomes demonstrated a pseudo-first-order kinetic degradation. Less degradation was observed at pH 3.4 and 4 degrees C. The degradation rate of calcitonin in solution, or in positively charged, negatively charged, and neutral liposomes, exhibited no significant difference. The particle size of the calcitonin-containing liposomes after storage for 1 month at pH 4.3 and 4 degrees C showed little change.     

Co-encapsulation of isoniazid and rifampicin in liposomes and characterization of liposomes by derivative spectroscopy

Taking into consideration the benefits of the combined therapy of isoniazid (INH) and rifampicin (RIF), this study focused on co-encapsulation of INH and RIF in the same liposome formulation. INH was incorporated in the aqueous phase and RIF in the lipid layer. Liposomes containing either INH or RIF were also prepared. All liposome formulations were compared for their loading capacity, encapsulation percentage and release properties. Drug amounts in the liposomes were estimated using peak-to-peak first-order derivative UV spectroscopy. Among the liposome formulations DPPC:chol liposomes showed the highest loading capacity (106.70 +/- 0.12 for INH and 18.17 +/- 0.06 (x 10(-3)) for RIF) and encapsulation percentage (73.84 +/- 0.78 for INH and 81.53 +/- 2.06 for RIF) compared to EPC:chol liposomes (loading capacity 93.36 +/- 0.58 for INH and 17.87 +/- 0.11 (x 10(-3)) for RIF; encapsulation percentage 64.61 +/- 0.51 for INH and 74.45 +/- 0.48 for RIF). Co-encapsulation of INH and RIF increased their individual encapsulation percentage and extended drug release compared to the formulations containing drug alone (Table 2). Results of this study support the conclusion that lipid and water soluble drugs can be successfully co-encapsulated in the same liposome formulation and also show that derivative UV spectroscopy is a sensitive method for direct and accurate quantification of these co-encapsulated drugs.     

Preparation of beta-artemether liposomes, their HPLC-UV evaluation and relevance for clearing recrudescent parasitaemia in Plasmodium chabaudi malaria-infected mice

Egg phosphatidylcholine-cholesterol liposome formulations containing the antimalarial drug beta-artemether have been prepared and analyzed for their encapsulating capacity, chemical stability, leakage, in vitro release and their therapeutic efficiency against Plasmodium chabaudi infection. A HPLC-UV analysis of beta-artemether liposomes without derivatisation was achieved. A good linearity of y=4437.7 x+469.01 (R(2)=0.9999) with a detection limit of 2 microg ml(-1) was reached. Prior to this, liposomal formulations composed of different molar ratios of EPC-CHOL were prepared to select beta-artemether crystal-free liposome preparations. The formulation corresponding to 4:3 and a total concentration of 300 mg lipids ml(-1) buffer (pH 7.2), which could incorporate as much as 1.5 mg beta-artemether was selected for therapy. A trapping efficiency of nearly 100% was reached, the drug being located in the lipid bilayers. A dialysis test demonstrated that the drug could be reversibly released from the liposomes, reaching equilibrium within 24 h. After 3 months storage at 4 degrees C, no leakage of beta-artemether had occurred indicating a high stability of the liposomes. These liposomes were used to treat mice infected with the virulent rodent malaria parasite Plasmodium chabaudi chabaudi, with a 100% cure by clearing the recrudescent parasitaemia.     

Characterization of calcitonin-containing liposome formulations for intranasal delivery

Calcitonin-containing liposome formulations were characterized to obtain information for evaluation of their feasibility in intranasal delivery. The parameters of liposomal charge characteristics, charge inducing agent concentration, calcitonin concentration and pH of the medium on the loading efficiency and leakage behaviour, and the chemical stability of calcitonin in liposomes were investigated. Results showed that the loading efficiency of calcitonin increased with increasing the added concentration of calcitonin. The magnitude of the loading efficiency due to the liposomal charge of negative, positive and neutral characteristics was in the order of negatively charged liposome &gt; neutral liposome &gt; positively charge liposome. The increase of molar ratio of phosphatidylserine in liposomes showed an increase of loading efficiency; while, the increase of molar ratios of stearylamine showed a decrease of loading efficiency. The loading efficiency at pH7.4 was greater than that at pH4.3. The leakage of positively charged liposomes was greater than that of neutral and negatively charged liposomes. The leakage at pH4.3 was faster than that at pH7.4. The leakage of positively charged liposomes increased as temperature increased. The chemical stability of calcitonin in both solution and liposomes demonstrated a pseudo-first-order kinetic degradation. Less degradation was observed at pH3.4 and 4°C. The degradation rate of calcitonin in solution, or in positively charged, negatively charged, and neutral liposomes, exhibited no significant difference. The particle size of the calcitonin-containing liposomes after storage for 1 month at pH4.3 and 4°C showed little change.     

pH-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system

The lack of stability in blood and the short blood circulation time of pH-sensitive liposomes are major drawbacks for their application in-vivo. To develop pH-sensitive, serum-stable and long-circulating liposomes as drug delivery systems, the impact of polyethylene glycol-derived phosphatidylethanolamine (DSPE-PEG) on the properties of pH-sensitive liposomes was investigated. pH-sensitive liposomes were prepared with dioleoylphosphatidylethanolamine (DOPE) and oleic acid (DOPE/oleic acid liposome) or DOPE and 1,2-dipalmitoylsuccinylglycerol (DOPE/DPSG liposome). The inclusion of DSPE-PEG enhanced the serum stability of both DOPE/oleic acid and DOPE/DPSG liposomes, but also shifted the pH-response curve of pH-sensitive liposomes to more acidic regions and reduced the maximum leakage percentage. The impact of DSPE-PEG, however, was much lower in the DOPE/DPSG liposomes than in the DOPE/oleic acid liposomes. In tumour tissue homogenates, where the pH is lower than normal healthy tissues, the pH-sensitive DOPE/DPSG liposomes released the entrapped markers rapidly, in comparison with pH-insensitive dipalmitoylphosphatidylcholine/cholesterol/DSPE-PEG liposomes. Moreover, the release rate was not affected by the content of DSPE-PEG. The blood circulation time of methotrexate incorporated in DOPE/UDPSG liposomes was significantly prolonged with increasing content of DSPE-PEG. Taken together, the liposomes composed of DOPE, DPSG and DSPE-PEG (up to 5%) were pH sensitive, plasma stable and had a long circulation time in the blood. The complete destabilization of the liposomes at tumour tissues suggests that the liposomes might be useful for the targeted delivery of drugs such as anticancer agents.     

Development of Pegylated Liposomal Vincristine Using Novel Sulfobutyl Ether Cyclodextrin Gradient: Is Improved Drug Retention Sufficient to Surpass DSPE–PEG-Induced Drug Leakage?

The purpose of this study is to develop novel stable PEGylated liposome vincristine formulations with optimal antitumor efficacy. Vincristine could interact with negatively charged distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), leading to rapid drug release from vesicles. To improve drug retention, vincristine was loaded into vesicles using sulfobutyl ether cyclodextrin (sbe-CD) as trapping agent. Despite that, vincristine could not form a precipitate with sbe-CD; the aggregation status of vincristine/sbe-CD inside vesicles must be complicated because drug retention was considerably improved in vivo. Theoretical consideration revealed that the release constant K equals to pA(m)k(1)k(2)/([H(+)](i)[sbe(-)](i)V(i) ), which can be used to expound why increasing drug/lipid ratio induced decreased vincristine circulation half-life. The stabilization effect afforded by sbe-CD was sufficient to surpass DSPE-PEG-induced drug leakage, so PEGylated liposomal vincristine formulations with prolonged circulation half-life (t(1/2): from 43.6 to 70.0 h) could be achieved, of which the formulation pLV-c-2.9-3 exhibited optimal antitumor effects and reduced toxicity. The strategy might be used to load other vinca alkaloids into PEGylated liposomes and improve their retention inside vesicles.     

Formulation of Liposome for topical delivery of arbutin

The aims of this study were to encapsulate arbutin (AR) in liposome to enhance the skin-whitening activity, and to investigate the effect of liposome formulation on the entrapment efficiency (EE%), skin permeation rate and skin deposition. The liposomes were prepared by a film dispersion method with several different formulations and were separated from the solution by using the gel-filtration method. The physical (size distribution, morphology) and chemical (drug entrapment efficiency, hairless mouse skin permeation and deposition) properties of liposomes were characterized. The entrapment efficiency in all liposome formulations varied between 4.35% and 17.63%, and was dependent on the lipid content. The particle sizes of liposomes were in the range of 179.9-212.8 nm in all liposome formulations. Although the permeation rate of AR in the liposome formulations decreased compared with AR solution, the deposition amount of AR in the epidermis/dermis layers increased in AR liposomal formulation. These results suggest that liposomal formulation could enhance the skin deposition of hydrophilic skin-whitening agents, thereby enhancing their activities.     

盐酸伊立替康脂质体的制备

目的结合脂质体这种新型给药载体的特征将盐酸伊利替康(Irinotecan,OPT-11)制备成脂质体以解决喜树碱类药物在生理条件下其结果中的内酯环易发生水解反应转变为羟酸盐形式,从而失去活性这个难问题.方法以包封率为主要评价指标,比较了传统的脂质体制备方法(薄膜分散法、乙醇注入法、反向蒸发法等)与主动载药方法-硫酸铵梯度法对伊立替康脂质体制备的影响.结果采用硫酸铵梯度法制备伊立替康脂质体可以获得较高的包封率、较大的药酯比.结论硫酸铵溶液的浓度、孵育时间和温度空白脂质体的粒径大小等是影响伊立替康脂质体包封率的主要因素.     

Oil-in-water liposomal emulsions: characterization and potential use in vaccine delivery

Emulsification of mineral oil by phospholipids donated by liposomes composed of dimyristoyl phosphatidylcholine, dimyristoyl phosphatidylglycerol, cholesterol, and lipid A by extrusion resulted in the formation of oil-in-water liposomal emulsions containing a substantial number of intact liposomes. Increasing the proportion of liposomes from 25 mM to 150 mM phospholipid and increasing the oil content from 2.5% (v/v) to 42.5% (v/v) changed the flow characteristics of the emulsions from fluid liquid-like to viscous. Likewise, the degree of stability of the emulsions was liposomal phospholipid concentration-dependent, ranging from partial emulsification in the range 25-100 mM to complete stabilization in the range 125-150 mM. Despite some loss of liposome integrity, as evidenced by the release of liposomal trapped glucose, emulsification of liposomes containing encapsulated prostate-specific antigen (PSA) exhibited antigen-specific immunostimulation in mice. These results suggest that liposomes containing encapsulated antigen can serve as constituents for the formulation of oil-in-water vaccines.     

Encapsulation of an antivasospastic drug, fasudil, into liposomes, and in vitro stability of the fasudil-loaded liposomes.

The objectives of this work were to develop a liposomal fasudil, an antivasospastic drug, as a possible means to deliver the encapsulated drug to the brain, and to characterize the stability of the liposomal formulation in vitro. Transmembrane electrochemical gradients of H+ or ammonium sulfate were created, and their effect on the uptake of fasudil into preformed hydrogenated soy phosphatidylcholine/cholesterol (HSPC/CHOL) liposomes were examined. Fasudil was successfully loaded into preformed liposomes in response to sulfate ion (SO4(2-)) and, in part, by H+. Encapsulation levels approaching 100% could be achieved up to a drug to lipid ratio of 0.364 (mol/mol). A stability study of the fasudil-loaded liposomes was performed by storage at 4 degrees C in 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffer (pH 7.4) and by incubation in human cerebrospinal fluid (CSF) at 37 degrees C. The formulations were stable with respect to drug retention as well as size alteration, for the period studied. A leakage study clearly showed the sustained release properties of the fasudil-loaded liposomes in human CSF. We recently reported that the intrathecal administration of liposomal fasudil significantly decreased ischemia, with no obvious adverse effect in a rat model [Neurol. Med. Chir. 41 (2001) 109]. Taken together, efficient encapsulation of fasudil into preformed liposomes, their long-term stability at 4 degrees C and the sustained release characteristics in CSF indicate that fasudil-loaded liposomes could be potential candidates for further clinical evaluation.     

Synthesis of cholesterol modified cationic lipids for liposomal drug delivery of antisense oligonucleotides.

The paper describes a novel synthesis of cholest-5-en-3 beta-yl-6-aminohexyl ether (AH-Chol). AH-Chol was used to prepare positively charged liposomes. The liposomes consisted of phospholipon 90H and the cationic cholesterol derivative in an equimolar ratio. Liposome preparation was achieved by membrane homogenization after rehydration of a dry lipid film. Oligonucleotides (ODN) were adsorbed to the cationic liposomes very efficiently. At an ODN/liposome ratio of 1:5 (10:50 micrograms/ml) 84.2 +/- 5.4% of the ODNs were bound to the liposomal membrane. Within the range of 1:40 and 1:100 charge neutralization occurred and the liposome dispersion showed an increase in particle size due to aggregation. Below or above this range of charge neutralization the ODN loaded liposome preparation was physically stable, no sedimentation, increase of vesicle size or vesicle aggregation occurred.     

Prolonged Blood Circulation of Methotrexate by Modulation of Liposomal Composition

Prolonged circulation by liposomal incorporation has been shown to enhance the therapeutic efficacy of drugs in many cases. The purpose of this study was to investigate whether the prolonged circulation of methotrexate (MTX) can be achieved by modulating the liposomal compositions. Various compositions of liposomes were prepared with 2:1 of phosphatidylcholine (PC) and cholesterol (CH) with or without distearoylphosphatidyl-ethanolamine-N-poly(ethyleneglycol) 2000 (DSPE-PEG). The MTX encapsulation efficiency depended on the type of PC used. It also appeared to increase by inclusion of DSPE-PEG. The size of liposomes decreased by the inclusion of DSPE-PEG. The inclusion of DSPE-PEG lowered the plasma-induced release of MTX from EggPC/CH and DPPC/CH liposomes, suggesting its enhancement effect on the liposomal stability. After intravenous injection to rats, the pharmaockinetics and biodistribution of MTX were significantly changed by liposomal incorporation and also by the composition of liposomes. The total body clearance of MTX incorporated in EggPC/CH, DPPC/CH, EggPC/CH/DSPE-PEG, and DPPC/CH/DSPE-PEG liposomes decreased 4.4-, 14.9-, 24.5-, and 53.1-fold, compared with that of free MTX. The ratio of MTX concentration in blood to liver and spleen after injection of DPPC/CH, EggPC/CH/DSPE-PEG, and DPPC/CH/DSPE-PEG liposomes was 5.4-, 8.5-, and 13.5-fold higher than that of EggPC/CH liposomes. Furthermore, the accumulation of MTX in the kidney, one of the organs in which MTX exhibits its toxicity, was significantly lowered by liposomal incorporation, especially by DSPE-PEG-containing liposomes. Taken together, DPPC/CH/DSPE-PEG liposomes most effectively prolonged the blood circulation, and reduced hepatosplenic and kidney uptake of MTX. DPPC/CH/DSPE-PEG liposomes may have potential as an efficient delivery system for MTX.     

Radiopaque liposomes: effect of formulation conditions on encapsulation efficiency

Liposomes containing sodium ioxitalamate were prepared by sonication. Suitable amounts of purified soybean phosphatidylcholine and cholesterol were used at various molar ratios. Stearylamine or dicetylphosphate were added to this lipid composition when charged liposomes were required. After sonication and removal of unencapsulated solute, this manufacturing process yielded small multilamellar vesicles as confirmed by electron microscopy. These liposomes did not exhibit a narrow range of size distribution; the mean particle size varied from 135 to 145 nm. With respect to the efficiency of encapsulation, two parameters were distinguishable: the volume of encapsulated aqueous space per unit of lipid weight and the percentage of the contrast agent added that became encapsulated in the liposomes. Investigation of the preparative parameters revealed that increased molar ratios of cholesterol yielded higher aqueous volume and iodine contents in the liposomes, which were attributed to a reduction of the liposome permeability to the contrast agent. However, the inclusion of cholesterol into the bilayer liposomal membrane was limited, probably by solubility restrictions. Negatively and positively charged liposomes had higher rates of encapsulation than did neutral liposomes. This result was expected since efficient encapsulation of polar compounds requires formation of large aqueous spaces within the vesicles per mole of lipids. Increase of the lipid fractions at a constant, reduced the aqueous volume entrapped per millimole of lipid and, consequently, the iodine content in the liposomes. However, an increase in the initial sodium ioxitalamate concentration diminished the aqueous volume entrapped in the liposomes but increased the iodine content.     

Encapsulation of vinorelbine into cholesterol-polyethylene glycol coated vesicles: drug loading and pharmacokinetic studies

Objectives Pegylated liposome formulations of vinorelbine with prolonged circulation half‐life (t½) are desirable. However, DSPE‐PEG could affect vinorelbine loading into vesicles due to electrostatic interactions. To resolve this problem, chol‐PEG was used to prepare pegylated liposomal vinorelbine and the factors affecting drug loading and plasma pharmacokinetics were investigated. Methods Vinorelbine was loaded into liposomes using a novel triethylamine 5‐sulfosalicylate gradient. The effects of cholesterol and chol‐PEG on drug loading were investigated. Pharmacokinetic studies were performed in normal KunMing mice treated with different liposomal vinorelbine formulations. To clarify the effects of chol‐PEG on membrane permeability, drug release experiments were performed based on the fluorescence dequenching phenomenon of a fluorescence marker. Key findings In contrast to DSPE‐PEG, even at high PEG grafting density (～8.3 mol%), chol‐PEG had no effect on vinorelbine loading into HSPC/cholesterol (3 : 1, mass ratio) vesicles. However, for the formulations with low cholesterol content (HSPC/cholesterol 4 : 1), loading efficiency decreased with increasing chol‐PEG content. In vivo, the vinorelbine t½ of low cholesterol formulations decreased with increasing chol‐PEG content, but for high cholesterol liposomes, the maximum vinorelbine t½ was achieved at ～3 mol% chol‐PEG grafting density. The resulting vinorelbine circulation t½ was ～9.47 h, which was greater than that of non‐pegylated liposomes (～5.55 h). Drug release experiments revealed that chol‐PEG might induce membrane defects and concomitant release of entrapped marker, especially at high chol‐PEG density. Conclusions Through the investigation of the effects of chol‐PEG and cholesterol, an optimum pegylated liposomal vinorelbine formulation with prolonged t½ was achieved. In plasma, the membrane defect induced by chol‐PEG may counteract the long circulation characteristics that chol‐PEG afforded. When these two opposite effects reached equilibrium, the maximum vinorelbine t½ was achieved.